Treating soft tissue via controlled drug release

ABSTRACT

Methods for inhibiting tissue ossification or calcification in a subject, comprising administering a therapeutically effective amount of BMP I inhibitor-loaded microparticles to a subject in need thereof, wherein the administration provides local and sustained release of the BMP I inhibitor thereby inhibiting tissue ossification or calcification.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.15/123,994, filed Sep. 6, 2016, which is the U.S. National Stage ofInternational Application No. PCT/US2015/019005, filed Mar. 5, 2015,which was published in English under PCT Article 21(2), which in turnclaims the benefit of U.S. Provisional Application No. 61/949,886, filedMar. 7, 2014, which is incorporated herein by reference in its entirety.

BACKGROUND

The surgical procedures currently used to treat local cartilage damage(focal lesions) are: microfracture (MFX), autologous chondrocyteimplantation (ACI), and mesenchymal stem cell (MSC) implantation.Although these procedures produce healthy cartilage, 18% of MFX and 16%of ACI require re-operation because of cartilage calcification, whilenearly all MSC treatments exhibit some extent of chondrocytehypertrophy, which is the initial stage of calcification.

Chondrocyte hypertrophy, the cell terminal differentiation stage, aswell as calcification in general, are the result of specific pathways,in particular of SMAD1/5/8 phosphorylation, a canonical BMP pathway.Recently, a small synthetic molecule, dorsomorphin, has been found tointerfere with SMAD1/5/8 phosphorylation, thus inhibiting chondrocyteterminal differentiation.

SUMMARY

Disclosed herein are methods for inhibiting tissue ossification orcalcification in a subject, comprising administering a therapeuticallyeffective amount of BMP I inhibitor-loaded microparticles to a subjectin need thereof, wherein the administration provides local and sustainedrelease of the BMP I inhibitor thereby inhibiting tissue ossification orcalcification.

Also disclosed herein are methods comprising administering atherapeutically effective amount of a BMP I inhibitor to a subject inneed of cartilage repair, wherein the BMP I inhibitor is locallydelivered at the site of the cartilage repair in a sustained releaseform, and the sustained release form comprises a BMP I inhibitor-loadedmicroparticles.

Further disclosed herein are methods for inhibiting tissue ossificationor calcification in a subject, comprising administering atherapeutically effective amount of inhibitor-loaded microparticles to asubject in need thereof, wherein the administration provides local andsustained release of the inhibitor thereby inhibiting tissueossification or calcification, and wherein the inhibitor interferes withactivation of SMAD 1/5/8.

Additionally disclosed herein are methods comprising administering atherapeutically effective amount of an inhibitor to a subject in need ofcartilage repair, wherein the inhibitor is locally delivered at the siteof the cartilage repair in a sustained release form, and the sustainedrelease form comprises inhibitor-loaded microparticles and the inhibitorinterferes with activation of SMAD 1/5/8.

Also disclosed herein is a therapeutic scaffold comprising (i) BMP Iinhibitor-loaded microparticles and (ii) chondrocytes, mesenchymal stemcells, or a combination thereof.

Further disclosed herein is a pharmaceutical composition comprising (i)BMP I inhibitor-loaded microparticles and (ii) at least onepharmaceutically acceptable carrier, adjuvant or excipient.

The foregoing will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of microsphere fabrication.

FIG. 2 shows the results of microsphere toxicity testing. Humanmesenchymal stem cells have been seeded at a density of 10,000/cm² on atissue culture treated six well plate with the addition of eitheralginate or PLGA microparticles. Alginate microparticles served as acontrol. After two days in culture with growth media (DMEM added withfetal bovine serum and penicillin/streptomycin/fungizone) cells weretested with a LIVE/DEAD® Viability/Cytotoxicity Kit to rapidlydiscriminates live from dead cells by simultaneously staining with thegreen-fluorescent calcein-AM and the red-fluorescent ethidiumhomodimer-1. The first indicates intracellular esterase activity, thelatter loss of integrity of the cell plasma membrane. Bright fieldimages are compared with green-fluorescent, red-fluorescent, and overlayimages. Microparticles appear as spheres in the bright field image (topleft of each panel), live cells are stained green in thegreen-fluorescent image (top right of each panel), dead cells arestained red in the red-fluorescent image (bottom left panel). Very fewred stains are visible for alginate microparticles. More are visible forcells treated with PLGA microparticles, however the red signal does notcome from the cells. By comparing the overlay images, it is clear thatmost of the red fluorescent co-localizes with PLGA microspheres. Thissuggests that ethidium homodimer-1 partitions into the PLGA polymer,which is not unexpected given the hydrophobic nature of ethidiumhomodimer-1. In conclusion, PLGA microparticles have minimal if noeffect on cell viability.

FIG. 3 is a graph depicting the results of metabolic activity ofmicrospheres tested with an MTS assay. This test included the samegroups as in FIG. 2 plus a positive control (cells seeded withoutmicroparticles) and a negative control (cells treated with methanol for45 minutes to ensure 100% cell death). All groups were tested withCellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) todetermine colorimetrically any change in the number of viable,metabolically active cells. Test was performed as per the assayprotocol. No significant change was observed in cells cultured witheither alginate or PLGA microparticles, with only a minor decrease inmetabolic signal for cells cultured with PLGA. This is a common featureusually ascribed to the slightly acidic degradation products of the PLGAmicroparticles. In conclusion, PLGA microparticles have minimal effecton cells metabolic activity.

FIG. 4 is a graph depicting dorsomorphin release. 10 mg of PLGAmicroparticles containing dorsomorphin have been suspended in 1 ml ofPBS. Each day, PBS is removed and stored at −20° C. until examinationand the microparticles are resuspended in 1 ml of PBS. Each experimentis repeated at least in triplicate. Periodically, the collected PBS isexamined by HPLC to quantify against a standard concentration curve theamount of dorsomorphin released. The graph reports the values of thecumulative release of dorsomorphin over a 20 day period in microgramsper milliliter for each 10 mg of microparticles.

FIG. 5 represents the outline for testing of preventing calcification.Experimental design to test the effects of triiodothyronine in inducingcalcification and of dorsomorphin in inhibiting calcification in humanosteochondral biopsies, an example of which is depicted on the right.

FIG. 6 is two graphs depicting ALP activity. The graphs represent thelevel of ALP activity in the osteochondral constructs media measuredwith a colorimetric ALP assay for the samples outlined in the previousfigure. The first graph reports a statistically significant (p<0.05)increase in ALP activity after treatment with triiodothyronine (T3)between day 0 and day 20. The second graph reports a statisticallysignificant (p<0.05) decrease in ALP activity between day 0 and day 20when samples are treated with both triiodothyronine and dorsomorphin.

FIGS. 7A-7F are microphotographs of human chondrocytes. Chondrocytesextracted from human donors have been cultured under a test medium toinduce hypertrophy for 14 or 21 days followed by 14 or 7 days ofchondrogenic medium, respectively. The test medium was eitherchondrogenic medium (negative control, no hypertrophy), mediumcontaining triiodothyronine (T3) (hypertrophic), osteogenic medium(hypertrophic).

FIGS. 8A-8G are graphs of gene expression. Human chondrocyte andosteoblasts were cultured for 28 days after seeding in athree-dimensional construct made of photocrosslinkable gelatin at aconcentration of 10 million cells/ml. Osteoblasts served as a positivecontrol. Chondrocytes constructs were cultured for 14, 21, or 28 days inchondrogenic or hypertrophic (containing T3) medium, followed by 14, 21,or 0 days, respectively, of culture in chondrogenic medium. Osteoblastsconstructs were cultured for 14, 21, or 28 days in osteogenic medium,followed by 14, 21, or 0 days, respectively, of culture in chondrogenicmedium. At the end of the 28 days test, all constructs were harvestedand RNA extracted to assess expression of chondrogenic genes (Col1a2,and Aggrecan) and of hypertrophic/osteogenic genes (ALP, COLX, MMP13,OCN).

FIG. 9 is a graph of bone volume determined by μCT in an in vivo mousemodel. Formation of ectopic bone nodules in the hind limbs of SCID miceinjected with muscle-derived multipotent progenitor cells (MPC) with orwithout BMP2 as compared to those injected with human foreskinfibroblasts (HFF) with or without BMP2, at 1, 2, 4 and 6 weeks.

FIG. 10 is microtopographs showing inhibition of muscle-derivedmultipotent progenitor cells (MPC) osteogenesis in 2D by 2.5 nM ofdorsomorphin (DM) as shown by alizarin red staining (magnification 10×).Osteogenesis was induced by addition of BMP to the medium.

FIGS. 11A and 11B are microphotographs of bone marrow-derivedmesenchymal stem cells (BMD-MSCs) showing inhibition of BMD-MSCosteogenesis in 2D culture by dorsomorphin (DM). Phase contrast imagesof alizarin red stained cultures treated with BMP, DM, and BMP+DM ascompared to control and DM cultures.

FIG. 12 is bone volume, determined by μCT, formed ectopically in thehind limbs of SCID mice injected with MPC+BMP2+/−dorsomorphin (DM). DMwas delivered in colution (sDM) or in micro particles (mpDM).Representative μCT 3D reconstructions of heterotopic ossification (HO)inhibition of BMP2-stimulated MPC osteogenesis in vivo by dorsomorphin.HO lesions in vivo following injection of MPC/BMP2 and (A) soluble (s)DM, (B) alone, and (C) DM in microparticles (no calcificationregistered). All scans were normalized to the device hydroxyapatitestandard (in vivo μCT 40; ScanCo).

DETAILED DESCRIPTION Terminology

The following explanations of terms and methods are provided to betterdescribe the present compounds, compositions and methods, and to guidethose of ordinary skill in the art in the practice of the presentdisclosure. It is also to be understood that the terminology used in thedisclosure is for the purpose of describing particular embodiments andexamples only and is not intended to be limiting.

An “animal” refers to living multi-cellular vertebrate organisms, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals. Similarly, the term “subject”includes both human and non-human subjects, including birds andnon-human mammals, such as non-human primates, companion animals (suchas dogs and cats), livestock (such as pigs, sheep, cows), as well asnon-domesticated animals, such as the big cats.

The term “co-administration” or “co-administering” refers toadministration of an agent disclosed herein with at least one othertherapeutic or diagnostic agent within the same general time period, anddoes not require administration at the same exact moment in time(although co-administration is inclusive of administering at the sameexact moment in time). Thus, co-administration may be on the same day oron different days, or in the same week or in different weeks. In certainembodiments, a plurality of therapeutic and/or diagnostic agents may beco-administered by encapsulating the agents within the microparticlesdisclosed herein.

“Inhibiting” refers to inhibiting the full development of a disease orcondition. “Inhibiting” also refers to any quantitative or qualitativereduction in biological activity or binding, relative to a control.

“Microparticle”, as used herein, unless otherwise specified, generallyrefers to a particle of a relatively small size, but not necessarily inthe micron size range. In certain embodiments, microparticlesspecifically refers to particles having a diameter from about 0.01 toabout 500 microns, preferably from about 1 to about 200 microns, morepreferably from about 5 to about 20 microns. As used herein, themicroparticle encompasses microspheres, microcapsules andmicroparticles, unless specified otherwise. A microparticle may be ofcomposite construction and is not necessarily a pure substance; it maybe spherical or any other shape.

Preventing” a disease or condition refers to prophylactic administeringa composition to a subject who does not exhibit signs of a disease orcondition for the purpose of decreasing the risk of developing apathology or condition, or diminishing the severity of a pathology orcondition.

A “therapeutically effective amount” refers to a quantity of a specifiedagent sufficient to achieve a desired effect in a subject being treatedwith that agent. Ideally, a therapeutically effective amount of an agentis an amount sufficient to inhibit or treat the disease or conditionwithout causing a substantial cytotoxic effect in the subject. Thetherapeutically effective amount of an agent will be dependent on thesubject being treated, the severity of the affliction, and the manner ofadministration of the therapeutic composition.

“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition after it has begun todevelop, or administering a compound or composition to a subject whoexhibits only early signs for the purpose of decreasing the risk ofdeveloping a pathology or condition, or diminishing the severity of apathology or condition. As used herein, the term “ameliorating,” withreference to a disease or pathological condition, refers to anyobservable beneficial effect of the treatment. The beneficial effect canbe evidenced, for example, by a delayed onset of clinical symptoms ofthe disease in a susceptible subject, a reduction in severity of some orall clinical symptoms of the disease, a slower progression of thedisease, an improvement in the overall health or well-being of thesubject, or by other parameters well known in the art that are specificto the particular disease. The phrase “treating a disease” also refersto inhibiting the full development of a disease.

“Pharmaceutical compositions” are compositions that include an amount(for example, a unit dosage) of one or more of the disclosed compoundstogether with one or more non-toxic pharmaceutically acceptableadditives, including carriers, diluents, and/or adjuvants, andoptionally other biologically active ingredients. Such pharmaceuticalcompositions can be prepared by standard pharmaceutical formulationtechniques such as those disclosed in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. (19th Edition).

The terms “pharmaceutically acceptable salt or ester” refers to salts oresters prepared by conventional means that include salts, e.g., ofinorganic and organic acids, including but not limited to hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonicacid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid,tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid,maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelicacid and the like. “Pharmaceutically acceptable salts” of the presentlydisclosed compounds also include those formed from cations such assodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and frombases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine,arginine, ornithine, choline, N,N′-dibenzylethylenediamine,chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine,diethylamine, piperazine, tris(hydroxymethyl)aminomethane, andtetramethylammonium hydroxide. These salts may be prepared by standardprocedures, for example by reacting the free acid with a suitableorganic or inorganic base. Any chemical compound recited in thisspecification may alternatively be administered as a pharmaceuticallyacceptable salt thereof. “Pharmaceutically acceptable salts” are alsoinclusive of the free acid, base, and zwitterionic forms. Descriptionsof suitable pharmaceutically acceptable salts can be found in Handbookof Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH(2002). When compounds disclosed herein include an acidic function suchas a carboxy group, then suitable pharmaceutically acceptable cationpairs for the carboxy group are well known to those skilled in the artand include alkaline, alkaline earth, ammonium, quaternary ammoniumcations and the like. Such salts are known to those of skill in the art.For additional examples of “pharmacologically acceptable salts,” seeBerge et al., J. Pharm. Sci. 66:1 (1977).

“Pharmaceutically acceptable esters” includes those derived fromcompounds described herein that are modified to include a carboxylgroup. An in vivo hydrolysable ester is an ester, which is hydrolysed inthe human or animal body to produce the parent acid or alcohol.Representative esters thus include carboxylic acid esters in which thenon-carbonyl moiety of the carboxylic acid portion of the ester groupingis selected from straight or branched chain alkyl (for example, methyl,n-propyl, t-butyl, or n-butyl), cycloalkyl, alkoxyalkyl (for example,methoxymethyl), aralkyl (for example benzyl), aryloxyalkyl (for example,phenoxymethyl), aryl (for example, phenyl, optionally substituted by,for example, halogen, C.sub.1-4 alkyl, or C.sub.1-4 alkoxy) or amino);sulphonate esters, such as alkyl- or aralkylsulphonyl (for example,methanesulphonyl); or amino acid esters (for example, L-valyl orL-isoleucyl). A “pharmaceutically acceptable ester” also includesinorganic esters such as mono-, di-, or tri-phosphate esters. In suchesters, unless otherwise specified, any alkyl moiety presentadvantageously contains from 1 to 18 carbon atoms, particularly from 1to 6 carbon atoms, more particularly from 1 to 4 carbon atoms. Anycycloalkyl moiety present in such esters advantageously contains from 3to 6 carbon atoms. Any aryl moiety present in such esters advantageouslycomprises a phenyl group, optionally substituted as shown in thedefinition of carbocycylyl above. Pharmaceutically acceptable estersthus include C₁-C₂₂ fatty acid esters, such as acetyl, t-butyl or longchain straight or branched unsaturated or omega-6 monounsaturated fattyacids such as palmoyl, stearoyl and the like. Alternative aryl orheteroaryl esters include benzoyl, pyridylmethyloyl and the like any ofwhich may be substituted, as defined in carbocyclyl above. Additionalpharmaceutically acceptable esters include aliphatic L-amino acid esterssuch as leucyl, isoleucyl and especially valyl.

For therapeutic use, salts of the compounds are those wherein thecounter-ion is pharmaceutically acceptable. However, salts of acids andbases which are non-pharmaceutically acceptable may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound.

The pharmaceutically acceptable acid and base addition salts asmentioned hereinabove are meant to comprise the therapeutically activenon-toxic acid and base addition salt forms which the compounds are ableto form. The pharmaceutically acceptable acid addition salts canconveniently be obtained by treating the base form with such appropriateacid. Appropriate acids comprise, for example, inorganic acids such ashydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric,nitric, phosphoric and the like acids; or organic acids such as, forexample, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e.ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic,fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric,methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic,cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.Conversely said salt forms can be converted by treatment with anappropriate base into the free base form.

The compounds containing an acidic proton may also be converted intotheir non-toxic metal or amine addition salt forms by treatment withappropriate organic and inorganic bases. Appropriate base salt formscomprise, for example, the ammonium salts, the alkali and earth alkalinemetal salts, e.g. the lithium, sodium, potassium, magnesium, calciumsalts and the like, salts with organic bases, e.g. the benzathine,N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids suchas, for example, arginine, lysine and the like.

The term “addition salt” as used hereinabove also comprises the solvateswhich the compounds described herein are able to form. Such solvates arefor example hydrates, alcoholates and the like.

The term “quaternary amine” as used hereinbefore defines the quaternaryammonium salts which the compounds are able to form by reaction betweena basic nitrogen of a compound and an appropriate quaternizing agent,such as, for example, an optionally substituted alkylhalide, arylhalideor arylalkylhalide, e.g. methyliodide or benzyliodide. Other reactantswith good leaving groups may also be used, such as alkyltrifluoromethanesulfonates, alkyl methanesulfonates, and alkylp-toluenesulfonates. A quaternary amine has a positively chargednitrogen. Pharmaceutically acceptable counterions include chloro, bromo,iodo, trifluoroacetate and acetate. The counterion of choice can beintroduced using ion exchange resins.

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbylC(O)—, preferably alkylC(O)—.

The term “acylamino” is art-recognized and refers to an amino groupsubstituted with an acyl group and may be represented, for example, bythe formula hydrocarbylC(O)NH—, preferably alkylC(O)NH—.

The term “acyloxy” is art-recognized and refers to a group representedby the general formula hydrocarbylC(O)O—, preferably alkylC(O)O—.

The term “aliphatic”, as used herein, includes straight, chained,branched or cyclic hydrocarbons which are completely saturated orcontain one or more units of unsaturation. Aliphatic groups may besubstituted or unsubstituted.

The term “alkoxy” refers to an oxygen having an alkyl group attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like.

The term “alkenyl”, as used herein, refers to an aliphatic groupcontaining at least one double bond and is intended to include both“unsubstituted alkenyls” and “substituted alkenyls”, the latter of whichrefers to alkenyl moieties having substituents replacing a hydrogen onone or more carbons of the alkenyl group. Such substituents may occur onone or more carbons that are included or not included in one or moredouble bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed below, except where stability isprohibitive. For example, substitution of alkenyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated. In preferred embodiments, a straight chain or branchedchain alkenyl has 1-12 carbons in its backbone, preferably 1-8 carbonsin its backbone, and more preferably 1-6 carbons in its backbone.Exemplary alkenyl groups include allyl, propenyl, butenyl,2-methyl-2-butenyl, and the like.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, and branched-chain alkyl groups.In preferred embodiments, a straight chain or branched chain alkyl has30 or fewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straightchains, C₃-C₃₀ for branched chains), and more preferably 20 or fewer. Incertain embodiments, alkyl groups are lower alkyl groups, e.g. methyl,ethyl, n-propyl, i-propyl, n-butyl and n-pentyl.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. In certain embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains). In preferred embodiments, the chain has ten or fewer carbon(C₁-C₁₀) atoms in its backbone. In other embodiments, the chain has sixor fewer carbon (C₁-C₆) atoms in its backbone.

Such substituents can include, for example, a halogen, a hydroxyl, acarbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl),a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),an alkoxyl, an alkylthio, an acyloxy, a phosphoryl, a phosphate, aphosphonate, an amino, an amido, an amidine, an imine, a cyano, a nitro,an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaryl or heteroaryl moiety.

The term “C_(x-y)” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups that contain from x to y carbons in the chain. Forexample, the term “C_(x-y)alkyl” refers to substituted or unsubstitutedsaturated hydrocarbon groups, including straight-chain alkyl andbranched-chain alkyl groups that contain from x to y carbons in thechain, including haloalkyl groups such as trifluoromethyl and2,2,2-tirfluoroethyl, etc. C₀ alkyl indicates a hydrogen where the groupis in a terminal position, a bond if internal. The terms “C₂-yalkenyl”and “C_(2-y)alkynyl” refer to substituted or unsubstituted unsaturatedaliphatic groups analogous in length and possible substitution to thealkyls described above, but that contain at least one double or triplebond respectively.

The term “alkylamino”, as used herein, refers to an amino groupsubstituted with at least one alkyl group.

The term “alkylthio”, as used herein, refers to a thiol groupsubstituted with an alkyl group and may be represented by the generalformula alkylS—.

The term “alkynyl”, as used herein, refers to an aliphatic groupcontaining at least one triple bond and is intended to include both“unsubstituted alkynyls” and “substituted alkynyls”, the latter of whichrefers to alkynyl moieties having substituents replacing a hydrogen onone or more carbons of the alkynyl group. Such substituents may occur onone or more carbons that are included or not included in one or moretriple bonds. Moreover, such substituents include all those contemplatedfor alkyl groups, as discussed above, except where stability isprohibitive. For example, substitution of alkynyl groups by one or morealkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups iscontemplated. In preferred embodiments, an alkynyl has 1-12 carbons inits backbone, preferably 1-8 carbons in its backbone, and morepreferably 1-6 carbons in its backbone. Exemplary alkynyl groups includepropynyl, butynyl, 3-methylpent-1-ynyl, and the like.

The term “amide”, as used herein, refers to a group

wherein R⁹ and R¹⁰ each independently represent a hydrogen orhydrocarbyl group, or R⁹ and R¹⁰ taken together with the N atom to whichthey are attached complete a heterocycle having from 4 to 8 atoms in thering structure.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein R⁹, R¹⁰, and R^(10′) each independently represent a hydrogen ora hydrocarbyl group, or R⁹ and R¹⁰ taken together with the N atom towhich they are attached complete a heterocycle having from 4 to 8 atomsin the ring structure.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith one or more aryl groups.

The term “aryl”, as used herein, include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 5- to 7-membered ring, more preferably a6-membered ring. Aryl groups include phenyl, phenol, aniline, and thelike.

The term “carbamate” is art-recognized and refers to a group

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbylgroup, such as an alkyl group.

The terms “carbocycle”, “carbocyclyl”, and “carbocyclic”, as usedherein, refers to a non-aromatic saturated or unsaturated ring in whicheach atom of the ring is carbon. Preferably a carbocycle ring containsfrom 3 to 10 atoms, more preferably from 5 to 7 atoms.

The term “carbocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The term “carbonate” is art-recognized and refers to a group —OCO₂—R⁹,wherein R⁹ represents a hydrocarbyl group, such as an alkyl group.

The term “carboxy”, as used herein, refers to a group represented by theformula —CO₂H.

The term “cycloalkyl”, as used herein, refers to the radical of asaturated aliphatic ring. In preferred embodiments, cycloalkyls havefrom 3-10 carbon atoms in their ring structure, and more preferably from5-7 carbon atoms in the ring structure. Suitable cycloalkyls includecycloheptyl, cyclohexyl, cyclopentyl, cyclobutyl and cyclopropyl.

The term “ester”, as used herein, refers to a group —C(O)OR⁹ wherein R⁹represents a hydrocarbyl group, such as an alkyl group or an aralkylgroup.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen”, as used herein, means halogen andincludes chloro, fluoro, bromo, and iodo.

The term “heteroalkyl”, as used herein, refers to a saturated orunsaturated chain of carbon atoms including at least one heteroatom(e.g., O, S, or NR⁵⁰, such as where R⁵⁰ is H or lower alkyl), wherein notwo hetero atoms are adjacent.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a hetaryl group.

The terms “heteroaryl” and “hetaryl” include substituted orunsubstituted aromatic single ring structures, preferably 5- to7-membered rings, more preferably 5- to 6-membered rings, whose ringstructures include at least one heteroatom (e.g., O, N, or S),preferably one to four or one to 3 heteroatoms, more preferably one ortwo heteroatoms. When two or more heteroatoms are present in aheteroaryl ring, they may be the same or different. The terms“heteroaryl” and “hetaryl” also include polycyclic ring systems havingtwo or more cyclic rings in which two or more carbons are common to twoadjoining rings wherein at least one of the rings is heteroaromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Preferredpolycyclic ring systems have two cyclic rings in which both of the ringsare aromatic. Heteroaryl groups include, for example, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine,pyridazine, quinoline, and pyrimidine, and the like.

The term “heteroatom”, as used herein, means an atom of any elementother than carbon or hydrogen. Preferred heteroatoms are nitrogen,oxygen, and sulfur.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. Heterocyclyl groupsinclude, for example, piperidine, piperazine, pyrrolidine, morpholine,lactones, lactams, and the like.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to behydrocarbyl for the purposes of this application, but substituents suchas acetyl (which has a ═O substituent on the linking carbon) and ethoxy(which is linked through oxygen, not carbon) are not. Hydrocarbyl groupsinclude, but are not limited to aryl, heteroaryl, carbocycle,heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are ten or fewer non-hydrogen atoms in thesubstituent, preferably six or fewer. A “lower alkyl”, for example,refers to an alkyl group that contains ten or fewer carbon atoms,preferably six or fewer. Examples of straight chain or branched chainlower alkyl include methyl, ethyl, isopropyl, propyl, butyl,tertiary-butyl, and the like. In certain embodiments, acyl, acyloxy,alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein arerespectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl,lower alkynyl, or lower alkoxy, whether they appear alone or incombination with other substituents, such as in the recitation aralkyl(in which case, for example, the atoms within the aryl group are notcounted when counting the carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Preferredpolycycles have 2-3 rings. Each of the rings of the polycycle can besubstituted or unsubstituted. In certain embodiments, each ring of thepolycycle contains from 3 to 10 atoms in the ring, preferably from 5 to7.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of the invention, the heteroatoms such as nitrogen may havehydrogen substituents and/or any permissible substituents of organiccompounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, an alkylthio,an acyloxy, a phosphoryl, a phosphate, a phosphonate, an amino, anamido, an amidine, an imine, a cyano, a nitro, an azido, a sulthydryl,an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or hetero aromaticmoiety.

Unless specifically stated as “unsubstituted,” references to chemicalmoieties herein are understood to include substituted variants. Forexample, reference to an “aryl” group or moiety implicitly includes bothsubstituted and unsubstituted variants.

The term “sulfate” is art-recognized and refers to the group —OSO₃H, ora pharmaceutically acceptable salt or ester thereof.

The term “sulfonamide” is art-recognized and refers to the grouprepresented by the general formulae

wherein R⁹ and R¹⁰ independently represents hydrogen or hydrocarbyl,such as alkyl.

The term “sulfoxide” is art-recognized and refers to the group —S(O)—R⁹,wherein R⁹ represents a hydrocarbyl, such as alkyl, aryl, or heteroaryl.

The term “sulfonate” is art-recognized and refers to the group —SO₃H, ora pharmaceutically acceptable salt or ester thereof.

The term “sulfone” is art-recognized and refers to the group —S(O)₂—R⁹,wherein R⁹ represents a hydrocarbyl, such as alkyl, aryl, or heteroaryl.

The term “thioester”, as used herein, refers to a group —C(O)SR⁹ or—SC(O)R⁹ wherein R⁹ represents a hydrocarbyl, such as alkyl.

The term “thioether”, as used herein, is equivalent to an ether, whereinthe oxygen is replaced with a sulfur.

The term “urea” is art-recognized and may be represented by the generalformula

wherein R⁹ and R¹⁰ independently represent hydrogen or a hydrocarbyl,such as alkyl.Overview

There is currently no method to prevent ossification of soft tissue. Inparticular, in clinical procedures of small defects cartilage repair,cartilage calcification can be a secondary, undesirable effect. Thesystems and methods disclosed herein would allow avoidance of cartilagecalcification. It could also be applied to a number of other instancesof calcification of other soft tissues (blood vessels, skin, kidney,tendons, etc.). It is designed to be a local therapy to minimize theissues otherwise related to systemic exposure.

Agents that inhibit bone morphogenetic protein (BMP) type I receptoractivity, particularly those that interfere with activation of BMPsignaling effectors SMAD 1/5/8, could be used in the local sustainedrelease delivery systems disclosed herein. Illustrative agents includedorsomorphin, LDN-193189 (Yu et al, Nature Medicine, 14:12, 1363-1369,2008), SB505124, noggin, cordin, and gremlin.

Illustrative inhibitors also include compounds represented by formula I:

wherein X is selected from CR¹⁵ and N; Y is selected from CR¹⁵ and N; Zis selected from CR³ and N; Ar is selected from substituted orunsubstituted aryl and heteroaryl, e.g., a six-membered ring, such asphenyl; L₁ is absent or selected from substituted or unsubstituted alkyland heteroalkyl; A and B, independently for each occurrence, areselected from CR¹⁶ and N, preferably CR¹⁶, e.g., CH; E and F,independently for each occurrence, are selected from CR⁵ and N,preferably CR⁵; preferably chosen such that no more than two of A, B, E,and F are N; R³ represents a substituent, e.g., selected from H andsubstituted or unsubstituted alkyl, heteroalkyl, cycloalkyl, halogen,hydroxyl, alkoxyl, alkylthio, acyloxy, acylamino, carbamate, cyano,sulfonyl, sulfoxido, sulfamoyl, or sulfonamido, e.g., lower alkyl; R⁴ isselected from substituted or unsubstituted alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, acyl, carboxyl,ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino, acylamino,carbamate, amido, amidino, sulfonyl, sulfoxido, sulfamoyl, orsulfonamido, e.g., substituted or unsubstituted alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl, heteroaryl, acyl, carboxyl, ester,acyloxy, amino, acylamino, carbamate, amido, amidino, sulfonyl,sulfoxido, sulfamoyl, or sulfonamido, preferably substituted orunsubstituted heterocyclyl or heteroaryl; R⁵, independently for eachoccurrence, represents a substituent, e.g., selected from H andsubstituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl,cycloalkylalkyl, heterocyclylalkyl, halogen, acyl, carboxyl, ester,hydroxyl, alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate,amido, amidino, cyano, sulfonyl, sulfoxido, sulfamoyl, or sulfonamido(preferably H or substituted or unsubstituted alkyl, alkenyl,heteroalkyl, halogen, acyl, carboxyl, ester, hydroxyl, alkoxyl,alkylthio, acyloxy, amino, acylamino, carbamate, amido, amidino, orcyano), or two occurrences of R⁵ taken together with the atoms to whichthey are attached form a substituted or unsubstituted 5- or 6-memberedcycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring, preferably anaryl or heteroaryl ring, e.g., a substituted or unsubstituted benzoring; R¹³ is absent or represents 1-2 substituents on the ring to whichit is attached and, independently for each occurrence, is selected fromsubstituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, halogen, hydroxyl,alkoxyl, alkylthio, acyloxy, acylamino, carbamate, cyano, sulfonyl,sulfoxido, sulfamoyl, or sulfonamido, preferably substituted orunsubstituted alkyl, heteroalkyl, halogen, hydroxyl, alkoxyl, alkylthio,acyloxy, acylamino, carbamate, or cyano; R¹⁵, independently for eachoccurrence, represents a substituent, e.g., selected from H andsubstituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, halogen, hydroxyl,alkoxyl, alkylthio, acyloxy, acylamino, carbamate, cyano, sulfonyl,sulfoxido, sulfamoyl, or sulfonamido, preferably H or substituted orunsubstituted alkyl, heteroalkyl, halogen, hydroxyl, alkoxyl, alkylthio,acyloxy, acylamino, carbamate, or cyano; R¹⁶, independently for eachoccurrence, represents a substituent, e.g., selected from H andsubstituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl,aralkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,cycloalkylalkyl, heterocyclylalkyl, halogen, acyl, carboxyl, ester,hydroxyl, alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate,amido, amidino, cyano, sulfonyl, sulfoxido, sulfamoyl, or sulfonamido,preferably H or substituted or unsubstituted alkyl, alkenyl,heteroalkyl, halogen, acyl, carboxyl, ester, hydroxyl, alkoxyl,alkylthio, acyloxy, amino, acylamino, carbamate, amido, or cyano, or apharmaceutically acceptable salt, ester, or prodrug thereof.

In certain embodiments, either Y is N or Ar comprises a nitrogen atom inthe ring.

In certain embodiments, E and F are each CR⁵, and both instances of R⁵together with the intervening atoms form a 5-, 6-, or 7-membered ringoptionally substituted by substituted or unsubstituted alkyl, alkenyl,alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, aralkyl,heteroaryl, heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl, halogen,acyl, carboxyl, ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino,acylamino, carbamate, amido, amidino, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamido (preferably substituted or unsubstitutedalkyl, alkenyl, heteroalkyl, halogen, acyl, carboxyl, ester, hydroxyl,alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate, amido,amidino, or cyano). In certain embodiments, E and F together form asubstituted or unsubstituted 6-membered cycloalkyl, heterocyclyl, arylor heteroaryl ring (e.g., a pyridine, piperidine, pyran, or piperazinering, etc.). In certain such embodiments, the ring comprises one to fouramine groups, while in other embodiments, the ring is a substituted orunsubstituted benzo ring (e.g.,

In certain such embodiments, the ring is substituted, e.g., byoptionally substituted alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkylalkyl,heterocyclylalkyl, halogen, acyl, carboxyl, ester, hydroxyl, alkoxyl,alkylthio, acyloxy, amino, acylamino, carbamate, amido, amidino, cyano,sulfonyl, sulfoxido, sulfamoyl, or sulfonamido (preferably alkyl,alkenyl, heteroalkyl, halogen, acyl, carboxyl, ester, hydroxyl, alkoxyl,alkylthio, acyloxy, amino, acylamino, carbamate, amido, amidino, orcyano).

In certain embodiments, Ar represents substituted or unsubstitutedheteroaryl e.g., pyrrole, furan, thiophene, imidazole, oxazole,thiazole, pyrazole, pyridine, pyrazine, pyridazine, quinoline, andpyrimidine, In certain embodiments, Ar represents substituted orunsubstituted aryl, such as phenyl. In certain embodiments, Ar is a6-membered ring, such as a phenyl ring, e.g., in which L₁ is disposed onthe para-position of Ar relative to the bicyclic core.

In certain embodiments as discussed above, substituents on Ar areselected from substituted or unsubstituted alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, aralkyl, heteroaryl,heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl, halogen, acyl,carboxyl, ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino,acylamino, carbamate, amido, amidino, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamido (preferably substituted or unsubstitutedalkyl, alkenyl, heteroalkyl, halogen, acyl, carboxyl, ester, hydroxyl,alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate, amido,amidino, or cyano).

In certain embodiments, L₁ represents a linker M_(k), wherein k is aninteger from 1-8, preferably from 2-4, and each M represents a unitselected from C(R¹⁸)₂, NR¹⁹, S, SO₂, or O, preferably selected so thatno two heteroatoms occur in adjacent positions, more preferably with atleast two carbon atoms between any nitrogen atom and another heteroatom;wherein R¹⁸, independently for each occurrence, is selected from H andsubstituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, hydroxyl, alkoxyl,alkylthio, acyloxy, amino, acylamino, carbamate, amido, amidino, cyano,sulfonyl, sulfoxido, sulfamoyl, or sulfonamido, preferably H or loweralkyl; and R¹⁹ is selected from H and substituted or unsubstitutedalkyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, oxide, amino,acylamino, carbamate, amido, amidino, sulfonyl, sulfamoyl, orsulfonamido, preferably H or lower alkyl.

In certain embodiments, L₁ is absent. In certain embodiments, L₁ isselected from substituted or unsubstituted alkyl (e.g., C₁-C₈ chains,preferably C₂-C₄ chains) and heteroalkyl. In certain such embodiments,L₁ has a structure

wherein n is an integer from 0 to 4, and Q is selected from CR¹⁰R¹¹NR¹²,S, S(O), and SO₂; R¹⁰ and R¹¹, independently for each occurrence, areselected from H and substituted or unsubstituted alkyl, heteroalkyl,cycloalkyl, heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, hydroxyl,alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate, amido,amidino, cyano, sulfonyl, sulfoxido, sulfamoyl, or sulfonamido,preferably H or lower alkyl; and R¹² is selected from H and substitutedor unsubstituted alkyl, cycloalkyl, heterocyclyl, heterocyclylalkyl,oxide, amino, acylamino, carbamate, amido, amidino, sulfonyl, sulfamoyl,or sulfonamido, preferably H or lower alkyl. In certain embodiments, L₁has a structure

wherein Q is CH₂, NH, S, SO₂, or O, preferably O.

In certain embodiments, R⁴ is

wherein R²¹, independently for each occurrence, is selected from H andsubstituted or unsubstituted alkyl, aralkyl, cycloalkyl, heterocyclyl,aryl, heteroaryl, heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl,acyl, sulfonyl, sulfamoyl, or sulfonamido, preferably H or lower alkyl.

In certain embodiments, R⁴ is heterocyclyl, e.g., comprising one or twoheteroatoms, such as N, S or O (e.g., piperidine, piperazine,pyrrolidine, morpholine, lactone, or lactam). In certain suchembodiments, R⁴ is heterocyclyl comprising one nitrogen atom, e.g.,piperidine or pyrrolidine, such as

wherein R²⁰ is absent or represents from 1-4 substituents on the ring towhich it is attached, e.g., selected from substituted or unsubstitutedalkyl, heteroaryl, aralkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl, acyl, hydroxyl,alkoxyl, alkylthio, acyloxy, sulfonyl, sulfoxido, sulfamoyl, andsulfonamido, preferably H or lower alkyl. In certain embodiments, R⁴ isheterocyclyl comprising two nitrogen atoms, e.g., piperazine. In certainembodiments, R⁴ is heterocyclyl comprising a nitrogen and an oxygenatom, e.g., morpholine.

In certain embodiments, R⁴ is a heterocyclyl or heteroaryl that includesan amine within the atoms of the ring, e.g., pyridyl, imidazolyl,pyrrolyl, piperidyl, pyrrolidyl, piperazyl, oxazolyl, isoxazolyl,thiazolyl, etc., and/or bears an amino substituent. In certainembodiments, R⁴ is

wherein R²⁰ is as defined above; W represents a bond or is selected fromC(R²¹)₂, O, or NR²¹; and R²¹, independently for each occurrence, isselected from H and substituted or unsubstituted alkyl, aralkyl,cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,cycloalkylalkyl, heterocyclylalkyl, acyl, sulfonyl, sulfamoyl, orsulfonamido, preferably H or lower alkyl.

In certain preferred embodiments, L₁ is absent and ArR⁴ has a structure

In certain embodiments as discussed above, substituents on R⁴ areselected from substituted or unsubstituted alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, aralkyl, heteroaryl,heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl, halogen, acyl,carboxyl, ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino,acylamino, carbamate, amido, amidino, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamido (preferably substituted or unsubstitutedalkyl, alkenyl, heteroalkyl, halogen, acyl, carboxyl, ester, hydroxyl,alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate, amido,amidino, or cyano).

In certain embodiments, L₁ is absent and R⁴ is directly attached to Ar.In embodiments wherein R⁴ is a six-membered ring directly attached to Arand bears an amino substituent at the 4-position of the ring relative toN.

In certain embodiments, L₁-R⁴ comprises a basic nitrogen-containinggroup, e.g., either L₁ comprises nitrogen-containing heteroalkyl or anamine-substituted alkyl, or R⁴ comprises a substituted or unsubstitutednitrogen-containing heterocyclyl or heteroaryl and/or is substitutedwith an amine substituent. In certain such embodiments, the pK_(a) ofthe conjugate acid of the basic nitrogen-containing group is 6 orhigher, or even 8 or higher.

In certain embodiments, L₁ has a structure

wherein n is an integer from 0 to 4, and R⁴ is heterocyclyl. In certainsuch embodiments, E and F together form a ring, e.g., a benzo ring,while in other embodiments, E and F do not form a ring.

In certain embodiments, L₁ is absent and R⁴ is heterocyclyl, especiallya nitrogen-containing heterocyclyl. In certain such embodiments, E and Ftogether form a ring, e.g., a benzo ring, while in other embodiments, Eand F do not form a ring. In certain embodiments, L₁ is absent and R⁴ ispiperidine, piperazine, pyrrolidine, or morpholine.

In certain of the embodiments disclosed above, if L₁ is alkyl orheteroalkyl and R⁴ is heterocyclyl, especially a nitrogen-containingheterocyclyl, then E and F together form a ring, e.g., a benzo ring. Incertain of the embodiments disclosed above, if L₁ has a structure

wherein n is an integer from 0 to 4 (especially from 1-2) and Q is S orO, then E and F together form a ring, e.g., a benzo ring.

In certain embodiments, either E and F are both CR⁵ and both occurrencesof R⁵ taken together with E and F form a ring, e.g., a benzo ring, or L₁is absent. In certain such embodiments, R⁴ is selected from substitutedor unsubstituted alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl,heteroaryl, acyl, carboxyl, ester, acyloxy, amino, acylamino, carbamate,amido, amidino, sulfonyl, sulfoxido, sulfamoyl, and sulfonamido. Incertain embodiments, either E and F are both CR⁵ and both occurrences ofR⁵ taken together with E and F form a ring, e.g., a benzo ring, or R⁴ isselected from substituted or unsubstituted cycloalkyl, aryl, heteroaryl,acyl, carboxyl, ester, acyloxy, amino, acylamino, carbamate, amido,amidino, sulfonyl, sulfoxido, sulfamoyl, and sulfonamido.

In certain of the embodiments disclosed above, if L₁ is absent, R⁴ iscycloalkyl or heterocyclyl (e.g., a nitrogen-containing heterocycle,such as piperidine, piperazine, pyrrolidine, morpholine, etc.).

In certain of the embodiments disclosed above, if L₁ is heteroalkyl andR⁴ is heterocyclyl (especially a nitrogen-containing heterocycle), thenY is CR¹⁵, wherein R¹⁵ is as defined above. In certain of theembodiments disclosed above, if L₁ is heteroalkyl and R⁴ is piperidine,then Y is CR¹⁵, wherein R¹⁵ is as defined above. In certain embodimentswherein Y is CR¹⁵, R¹⁵ is selected from H, lower alkyl, heteroalkyl, andester (e.g., lower alkyl ester, such as methyl ester).

In certain of the embodiments disclosed above, if L₁ is heteroalkyl andR⁴ is heterocyclyl (especially nitrogen-containing heterocyclyl), then Xis CR¹⁵, wherein R¹⁵ is as defined above. In certain of the embodimentsdisclosed above, if L₁ is heteroalkyl and R⁴ is piperidine, then X isCR¹⁵, wherein R¹⁵ is as defined above. In certain embodiments wherein Xis R¹⁵, R¹⁵ is selected from H, lower alkyl, and hetero alkyl.

In certain of the embodiments disclosed above, if L₁ is heteroalkyl andR⁴ is heterocyclyl (especially nitrogen-containing heterocyclyl), then Zis CR³, wherein R³ is as defined above. In certain of the embodimentsdisclosed above, if L₁ is heteroalkyl and R⁴ is piperidine, then Z isCR³, wherein R³ is as defined above. In certain embodiments wherein Z isCR³, R³ is selected from H, lower alkyl, and hetero alkyl.

In certain of the embodiments disclosed above, if L₁ is heteroalkyl andR⁴ is heterocyclyl (especially a nitrogen-containing heterocycle, suchas piperidine), R¹³ represents 2 substituents on the ring to which it isattached and, independently for each occurrence, is selected fromsubstituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, halogen, hydroxyl,alkoxyl, alkylthio, acyloxy, acylamino, carbamate, cyano, sulfonyl,sulfoxido, sulfamoyl, or sulfonamido.

In certain of the embodiments disclosed above, if L₁ is heteroalkyl andR⁴ is heterocyclyl (especially a nitrogen-containing heterocycle, suchas piperidine), Ar represents substituted or unsubstituted heteroaryl(e.g., pyrrole, furan, thiophene, imidazole, oxazole, thiazole,pyrazole, pyridine, pyrazine, pyridazine, quinoline, and pyrimidine). Incertain such embodiments, Ar is substituted with one or moresubstituents selected from alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl,cycloalkylalkyl, heterocyclylalkyl, halogen, acyl, carboxyl, ester,hydroxyl, alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate,amido, amidino, cyano, sulfonyl, sulfoxido, sulfamoyl, or sulfonamido.

In certain of the embodiments disclosed above, if L₁ is heteroalkyl andR⁴ is heterocyclyl (e.g., piperidine, piperazine, pyrrolidine,morpholine, lactones, lactams, and the like), R⁴ is substituted with oneor more substituents selected from alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl,cycloalkylalkyl, heterocyclylalkyl, halogen, acyl, carboxyl, ester,hydroxyl, alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate,amido, amidino, cyano, sulfonyl, sulfoxido, sulfamoyl, or sulfonamido.

In certain of the embodiments disclosed above, compounds have one ormore of the following features:

either Y is N or Ar comprises a nitrogen atom in the ring;

L₁ is absent;

E and F together form a ring;

R⁴ is cycloalkyl, aryl, or heteroaryl;

X is CR¹⁵;

Y is CR¹⁵;

Z is CR³;

R¹³ represents 1-2 substituents on the ring to which it is attached and,independently for each occurrence, is selected from substituted orunsubstituted alkyl, heteroalkyl, cycloalkyl, heterocyclyl,cycloalkylalkyl, heterocyclylalkyl, halogen, hydroxyl, alkoxyl,alkylthio, acyloxy, acylamino, carbamate, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamido;

Ar represents substituted or unsubstituted heteroaryl (e.g., pyrrole,furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine,pyrazine, pyridazine, quinoline, and pyrimidine);

Ar is substituted with one or more substituents selected from alkyl,alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, aralkyl,heteroaryl, heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl, halogen,acyl, carboxyl, ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino,acylamino, carbamate, amido, amidino, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamido; and

R⁴ is substituted with one or more substituents selected from alkyl,alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, aralkyl,heteroaryl, heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl, halogen,acyl, carboxyl, ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino,acylamino, carbamate, amido, amidino, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamido.

In one aspect, the compounds that inhibit BMP-induced phosphorylation ofSMAD1/5/8 including compounds represented by general formula II

wherein X is selected from CR₁₅ and N; Y is selected from CR₁₅ and N; Zis selected from CR₃ and N; Ar is selected from substituted orunsubstituted aryl and heteroaryl, e.g., a six-membered ring, such asphenyl; L₁ is absent or selected from substituted or unsubstituted alkyland heteroalkyl; Py is substituted or unsubstituted 4-pyridinyl or4-quinolinyl, e.g., optionally substituted with substituted orunsubstituted alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, heteroaralkyl, cycloalkylalkyl,heterocyclylalkyl, halogen, acyl, carboxyl, ester, amino, acylamino,carbamate, amido, amidino, cyano, sulfonyl, sulfoxido, sulfamoyl, orsulfonamido; and R³ represents a substituent, e.g., selected from H andsubstituted or unsubstituted alkyl, heteroalkyl, cycloalkyl, halogen,hydroxyl, alkoxyl, alkylthio, acyloxy, acylamino, carbamate, cyano,sulfonyl, sulfoxido, sulfamoyl, or sulfonamido, e.g., lower alkyl; R⁴ isselected from substituted or unsubstituted alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, acyl, carboxyl,ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino, acylamino,carbamate, amido, amidino, sulfonyl, sulfoxido, sulfamoyl, orsulfonamido, e.g., substituted or unsubstituted alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl, heteroaryl, acyl, carboxyl, ester,acyloxy, amino, acylamino, carbamate, amido, amidino, sulfonyl,sulfoxido, sulfamoyl, or sulfonamido, preferably substituted orunsubstituted heterocyclyl or heteroaryl; R⁵, independently for eachoccurrence, represents a substituent, e.g., selected from H andsubstituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl,cycloalkylalkyl, heterocyclylalkyl, halogen, acyl, carboxyl, ester,hydroxyl, alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate,amido, amidino, cyano, sulfonyl, sulfoxido, sulfamoyl, or sulfonamido(preferably H or substituted or unsubstituted alkyl, alkenyl,heteroalkyl, halogen, acyl, carboxyl, ester, hydroxyl, alkoxyl,alkylthio, acyloxy, amino, acylamino, carbamate, amido, amidino, orcyano), or two occurrences of R³ taken together with the atoms to whichthey are attached form a substituted or unsubstituted 5- or 6-memberedcycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring, preferably anaryl or heteroaryl ring, e.g., a substituted or unsubstituted benzoring; R¹³ is absent or represents 1-2 substituents on the ring to whichit is attached and, independently for each occurrence, is selected fromsubstituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, halogen, hydroxyl,alkoxyl, alkylthio, acyloxy, acylamino, carbamate, cyano, sulfonyl,sulfoxido, sulfamoyl, or sulfonamido, preferably substituted orunsubstituted alkyl, heteroalkyl, halogen, hydroxyl, alkoxyl, alkylthio,acyloxy, acylamino, carbamate, or cyano; R¹⁵, independently for eachoccurrence, represents a substituent, e.g., selected from H andsubstituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, halogen, hydroxyl,alkoxyl, alkylthio, acyloxy, acylamino, carbamate, cyano, sulfonyl,sulfoxido, sulfamoyl, or sulfonamido, preferably H or substituted orunsubstituted alkyl, heteroalkyl, halogen, hydroxyl, alkoxyl, alkylthio,acyloxy, acylamino, carbamate, or cyano; R¹⁶, independently for eachoccurrence, represents a substituent, e.g., selected from H andsubstituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl,aralkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,cycloalkylalkyl, heterocyclylalkyl, halogen, acyl, carboxyl, ester,hydroxyl, alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate,amido, amidino, cyano, sulfonyl, sulfoxido, sulfamoyl, or sulfonamido,preferably H or substituted or unsubstituted alkyl, alkenyl,heteroalkyl, halogen, acyl, carboxyl, ester, hydroxyl, alkoxyl,alkylthio, acyloxy, amino, acylamino, carbamate, amido, or cyano, or apharmaceutically acceptable salt, ester, or prodrug thereof.

In certain embodiments, either Y is N or Ar comprises a nitrogen atom inthe ring.

In certain embodiments, Py is substituted by substituted orunsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkylalkyl,heterocyclylalkyl, halogen, acyl, carboxyl, ester, hydroxyl, alkoxyl,alkylthio, acyloxy, amino, acylamino, carbamate, amido, amidino, cyano,sulfonyl, sulfoxido, sulfamoyl, or sulfonamido (preferably substitutedor unsubstituted alkyl, alkenyl, heteroalkyl, halogen, acyl, carboxyl,ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino, acylamino,carbamate, amido, amidino, or cyano).

In certain embodiments, Ar represents substituted or unsubstitutedheteroaryl e.g., pyrrole, furan, thiophene, imidazole, oxazole,thiazole, pyrazole, pyridine, pyrazine, pyridazine, quinoline, andpyrimidine. In certain embodiments, Ar represents substituted orunsubstituted aryl, such as phenyl. In certain embodiments, Ar is a6-membered ring, such as a phenyl ring, e.g., in which L₁ is disposed onthe para-position of Ar relative to the bicyclic core.

In certain embodiments as discussed above, substituents on Ar areselected from substituted or unsubstituted alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, aralkyl, heteroaryl,heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl, halogen, acyl,carboxyl, ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino,acylamino, carbamate, amido, amidino, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamido (preferably substituted or unsubstitutedalkyl, alkenyl, heteroalkyl, halogen, acyl, carboxyl, ester, hydroxyl,alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate, amido,amidino, or cyano).

In certain embodiments, L₁ represents a linker M_(k), wherein k is aninteger from 1-8, preferably from 2-4, and each M represents a unitselected from C(R¹⁸)₂, NR¹⁹, S, SO₂, or O, preferably selected so thatno two heteroatoms occur in adjacent positions, more preferably with atleast two carbon atoms between any nitrogen atom and another heteroatom;wherein R¹⁸, independently for each occurrence, is selected from H andsubstituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, hydroxyl, alkoxyl,alkylthio, acyloxy, amino, acylamino, carbamate, amido, amidino, cyano,sulfonyl, sulfoxido, sulfamoyl, or sulfonamido, preferably H or loweralkyl; and R¹⁹ is selected from H and substituted or unsubstitutedalkyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, oxide, amino,acylamino, carbamate, amido, amidino, sulfonyl, sulfamoyl, orsulfonamido, preferably H or lower alkyl.

In certain embodiments, L₁ is absent. In certain embodiments, L₁ isselected from substituted or unsubstituted alkyl (e.g., C₁-C₈ chains,preferably C₂-C₄ chains) and heteroalkyl. In certain such embodiments,L₁ has a structure

wherein n is an integer from 0 to 4, and Q is selected from CR¹⁰R¹¹,NR¹², S, S(O), and SO₂; R¹⁰ and R¹¹, independently for each occurrence,are selected from H and substituted or unsubstituted alkyl, heteroalkyl,cycloalkyl, heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, hydroxyl,alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate, amido,amidino, cyano, sulfonyl, sulfoxido, sulfamoyl, or sulfonamido,preferably H or lower alkyl; and R¹² is selected from H and substitutedor unsubstituted alkyl, cycloalkyl, heterocyclyl, heterocyclylalkyl,oxide, amino, acylamino, carbamate, amido, amidino, sulfonyl, sulfamoyl,or sulfonamido, preferably H or lower alkyl. In certain embodiments, L₁has a structure

wherein Q is CH.sub.2, NH, S, SO.sub.2, or O, preferably O.In certain embodiments, R⁴ is

wherein R²¹, independently for each occurrence, is selected from H andsubstituted or unsubstituted alkyl, aralkyl, cycloalkyl, heterocyclyl,aryl, heteroaryl, heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl,acyl, sulfonyl, sulfamoyl, or sulfonamido, preferably H or lower alkyl.

In certain embodiments, R⁴ is heterocyclyl, e.g., comprising one or twoheteroatoms, such as N, S or O (e.g., piperidine, piperazine,pyrrolidine, morpholine, lactone, or lactam). In certain suchembodiments, R⁴ is heterocyclyl comprising one nitrogen atom, e.g.,piperidine or pyrrolidine, such as

wherein R²⁰ is absent or represents from 1-4 substituents on the ring towhich it is attached, e.g., selected from substituted or unsubstitutedalkyl, heteroaryl, aralkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl, acyl, hydroxyl,alkoxyl, alkylthio, acyloxy, sulfonyl, sulfoxido, sulfamoyl, andsulfonamido, preferably H or lower alkyl. In certain embodiments, R⁴ isheterocyclyl comprising two nitrogen atoms, e.g., piperazine. In certainembodiments, R⁴ is heterocyclyl comprising a nitrogen and an oxygenatom, e.g., morpholine.

In certain embodiments, R⁴ is a heterocyclyl or heteroaryl that includesan amine within the atoms of the ring, e.g., pyridyl, imidazolyl,pyrrolyl, piperidyl, pyrrolidyl, piperazyl, oxazolyl, isoxazolyl,thiazolyl, etc., and/or bears an amino substituent. In certainembodiments, R⁴ is

wherein R²⁰ is as defined above; W represents a bond or is selected fromC(R²¹)₂, O, or NR²¹; and R²¹, independently for each occurrence, isselected from H and substituted or unsubstituted alkyl, aralkyl,cycloalkyl, heterocyclyl, aryl, heteroaryl, heteroaralkyl,cycloalkylalkyl, heterocyclylalkyl, acyl, sulfonyl, sulfamoyl, orsulfonamido, preferably H or lower alkyl.

In certain embodiments as discussed above, substituents on R⁴ areselected from substituted or unsubstituted alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, aralkyl, heteroaryl,heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl, halogen, acyl,carboxyl, ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino,acylamino, carbamate, amido, amidino, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamido (preferably substituted or unsubstitutedalkyl, alkenyl, heteroalkyl, halogen, acyl, carboxyl, ester, hydroxyl,alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate, amido,amidino, or cyano).

In certain embodiments, L₁ is absent and R⁴ is directly attached to Ar.In embodiments wherein R⁴ is a six-membered ring directly attached to Arand bears an amino substituent at the 4-position of the ring relative toN, the N and amine substituents may be disposed trans on the ring.

In certain embodiments, L₁-R⁴ comprises a basic nitrogen-containinggroup, e.g., either L₁ comprises nitrogen-containing heteroalkyl or anamine-substituted alkyl, or R⁴ comprises a substituted or unsubstitutednitrogen-containing heterocyclyl or heteroaryl and/or is substitutedwith an amine substituent. In certain such embodiments, the pK_(a) ofthe conjugate acid of the basic nitrogen-containing group is 6 orhigher, or even 8 or higher.

In certain embodiments, L₁ has a structure

wherein n is an integer from 0 to 4, and R⁴ is heterocyclyl. In certainsuch embodiments, Py is 4-quinolinyl, while in other embodiments, Py is4-pyridinyl.

In certain embodiments, L₁ is absent and R⁴ is heterocyclyl, especiallya nitrogen-containing heterocyclyl. In certain such embodiments, Py is4-quinolinyl, while in other embodiments, Py is 4-pyridinyl. In certainembodiments, L₁ is absent and R⁴ is piperidine, piperazine, pyrrolidine,or morpholine.

In certain of the embodiments disclosed above, if L₁ is alkyl orheteroalkyl and R⁴ is heterocyclyl, especially a nitrogen-containingheterocyclyl, then Py is 4-quinolinyl. In certain of the embodimentsdisclosed above, if L₁ has a structure

wherein n is an integer from 0 to 4 (especially from 1-2) and Q is S orO, then Py is 4-quinolinyl.

In certain embodiments, either Py is 4-quinolinyl, or L₁ is absent. Incertain such embodiments, R⁴ is selected from substituted orunsubstituted alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl,heteroaryl, acyl, carboxyl, ester, acyloxy, amino, acylamino, carbamate,amido, amidino, sulfonyl, sulfoxido, sulfamoyl, and sulfonamido. Incertain embodiments, either Py is 4-quinolinyl, or R⁴ is selected fromsubstituted or unsubstituted cycloalkyl, aryl, heteroaryl, acyl,carboxyl, ester, acyloxy, amino, acylamino, carbamate, amido, amidino,sulfonyl, sulfoxido, sulfamoyl, and sulfonamido.

In certain of the embodiments disclosed above, if L₁ is absent, R⁴ iscycloalkyl or heterocyclyl (e.g., a nitrogen-containing heterocycle,such as piperidine, piperazine, pyrrolidine, morpholine, etc.).

In certain of the embodiments disclosed above, if L₁ is heteroalkyl andR⁴ is heterocyclyl (especially a nitrogen-containing heterocycle), thenY is CR¹⁵, wherein R¹⁵ is as defined above. In certain of theembodiments disclosed above, if L₁ is heteroalkyl and R⁴ is piperidine,then Y is CR¹⁵, wherein R¹⁵ is as defined above. In certain embodimentswherein Y is CR¹⁵, R¹⁵, is selected from H, lower alkyl, heteroalkyl,and ester (e.g., lower alkyl ester, such as methyl ester).

In certain of the embodiments disclosed above, if L₁ is heteroalkyl andR⁴ is heterocyclyl (especially nitrogen-containing heterocyclyl), then Xis CR¹⁵, wherein R¹⁵ is as defined above. In certain of the embodimentsdisclosed above, if L₁ is heteroalkyl and R⁴ is piperidine, then X isCR¹⁵, wherein R¹⁵ is as defined above. In certain embodiments wherein Xis R¹⁵, R¹⁵ is selected from H, lower alkyl, and hetero alkyl.

In certain of the embodiments disclosed above, if L₁ is heteroalkyl andR⁴ is heterocyclyl (especially nitrogen-containing heterocyclyl), Z isCR³, wherein R³ is as defined above. In certain of the embodimentsdisclosed above, if L₁ is heteroalkyl and R⁴ is piperidine, then Z isCR³, wherein R³ is as defined above. In certain embodiments wherein Z isCR³, R³ is selected from H, lower alkyl, and heteroalkyl.

In certain of the embodiments disclosed above, if L₁ is heteroalkyl andR⁴ is heterocyclyl (especially a nitrogen-containing heterocycle, suchas piperidine), R¹³ represents 2 substituents on the ring to which it isattached and, independently for each occurrence, is selected fromsubstituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, halogen, hydroxyl,alkoxyl, alkylthio, acyloxy, acylamino, carbamate, cyano, sulfonyl,sulfoxido, sulfamoyl, or sulfonamido.

In certain of the embodiments disclosed above, if L₁ is heteroalkyl andR⁴ is heterocyclyl (especially a nitrogen-containing heterocycle, suchas piperidine), Ar represents substituted or unsubstituted heteroaryl(e.g., pyrrole, furan, thiophene, imidazole, oxazole, thiazole,pyrazole, pyridine, pyrazine, pyridazine, quinoline, and pyrimidine). Incertain such embodiments, Ar is substituted with one or moresubstituents selected from alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl,cycloalkylalkyl, heterocyclylalkyl, halogen, acyl, carboxyl, ester,hydroxyl, alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate,amido, amidino, cyano, sulfonyl, sulfoxido, sulfamoyl, or sulfonamido.

In certain of the embodiments disclosed above, if L₁ is heteroalkyl andR⁴ is heterocyclyl (e.g., piperidine, piperazine, pyrrolidine,morpholine, lactones, lactams, and the like), R⁴ is substituted with oneor more substituents selected from alkyl, alkenyl, alkynyl, heteroalkyl,cycloalkyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl,cycloalkylalkyl, heterocyclylalkyl, halogen, acyl, carboxyl, ester,hydroxyl, alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate,amido, amidino, cyano, sulfonyl, sulfoxido, sulfamoyl, or sulfonamido.

In certain of the embodiments disclosed above, compounds have one ormore of the following features:

either Y is N or Ar comprises a nitrogen atom in the ring;

L₁ is absent;

Py is 4-quinolinyl;

R₄ is cycloalkyl, aryl, or heteroaryl;

X is CR¹⁵;

Y is CR¹⁵;

Z is CR³;

R¹³ represents 1-2 substituents on the ring to which it is attached and,independently for each occurrence, is selected from substituted orunsubstituted alkyl, heteroalkyl, cycloalkyl, heterocyclyl,cycloalkylalkyl, heterocyclylalkyl, halogen, hydroxyl, alkoxyl,alkylthio, acyloxy, acylamino, carbamate, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamido;

Ar represents substituted or unsubstituted heteroaryl (e.g., pyrrole,furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine,pyrazine, pyridazine, quinoline, and pyrimidine);

Ar is substituted with one or more substituents selected from alkyl,alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, aralkyl,heteroaryl, heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl, halogen,acyl, carboxyl, ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino,acylamino, carbamate, amido, amidino, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamido; and

R⁴ is substituted with one or more substituents selected from alkyl,alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, aralkyl,heteroaryl, heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl, halogen,acyl, carboxyl, ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino,acylamino, carbamate, amido, amidino, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamide.

Illustrative compounds of Formula I and Formula II include:

The compounds of Formula I and Formula II are disclosed in US2011/0053930 A1.

These agents could be applied to prevent cartilage calcification afterclinical repair procedures, for example knee cartilage repair. In orderto use these agents to prevent calcification in the repair of a focallesion, a prolonged and local exposure to these agents would berequired. Disclosed herein is a system capable of a localized andsustained release.

The system is based on polymer microparticles, such as PLGAmicroparticles, in which particle preparation parameters are designed tomodify the release profile of the BMP I inhibitors. Such microparticles(MPs) may be incorporated in the clotting blood during microfracture, orwithin a scaffold seeded with chondrocytes or MSCs, for highly localizedlocal and sustained delivery of the BMP I inhibitor to specificallyblock chondrocyte terminal differentiation.

Dorsomorphin has been encapsulated in PLGA-based MPs of micrometer scaledimensions. As the particles degrade, dorsomorphin is releasedgenerating a local concentration within the range required to preventlocal calcification. To confirm this, human osteochondral plugs (3 mm by3 mm by 8 mm of a cartilage and bone biopsy-like sample) have beenforced to calcify using triiodothyronine (T3) and then exposed todorsomorphin. A calcification assay (ALP assay) and real time-PCRconfirmed that the dorsomorphin concentration arrested the calcificationprocess.

Applications for the systems disclosed herein for avoiding undesiredcalcifications are numerous. Ectopic calcification, i.e., thebiomineralization of soft tissues that would normally not ossify,affects a variety of tissues, from vasculature, to tendons/ligaments, tomuscle tissues and invasive surgeries are often required to remove thecalcified tissue. Calcification may also be an issue in regenerativemedicine when engineered constructs are implanted to substitute damagedtissue but they start to ossify instead.

Local delivery of BMP I receptor inhibitors could play a key role inimproving current reparative therapies. Specifically, in cartilagerepair procedures the formation of calcified cartilage or osteophytesmay be a significant issue requiring follow up surgery that could beavoided by the systems disclosed herein. The systems could also haveadditional application in pathologies in which ectopic calcification orheterotopic ossification affects other tissues (skin, kidney, bloodvessels, hearth, tendons/ligaments, muscles, etc.), for instancedegenerative calcific aortic stenosis that is one of the most commonvalvular lesion encountered in clinical cardiology. A further useincludes inhibition of the formation of intralesional osteophytes.

In certain embodiments the loaded microparticles may be used fortreating a subject suffering from or susceptible to heterotopicossification conditions. The subject may have been diagnosed with, ordetermined to be suffering from, an heterotopic ossification condition.In certain embodiments, the subject may have active, ongoing, tissueossification or calcification.

For example, the loaded microparticles disclosed herein may also be usedfor treating traumatic brain injury (particularly treating or preventingheterotopic ossification in the elbow, shoulder and/or hip aftertraumatic brain injury), cerebrovascular accident, paraplegia orquadriplegia, heterotopic ossification in the hip after spinal injury,poliomyelitis, Guillain-Barre syndrome, muscle hematoma, jointdislocation, post-hip or knee arthroplasty (particularly contiguous withor near to the hip after hip replacement), surgical scars (particularlyabdominal scars after surgery), severe burns (particularly treating orpreventing heterotopic ossification in the elbow and/or other jointsafter severe burns), secondary osteoma cutis, atherosclerosis(particularly treating or preventing heterotopic ossification incoronary arteries in atherosclerosis), valvular heat disease(particularly treating or preventing heterotopic ossification followingimplantation of a heart valve substitute), traumatic amputations(particularly treating or preventing heterotopic ossification in muscleafter a traumatic amputation), ankylosing spondylitis, psoriaticarthritis, osteoarthritis (particularly osteoarthritic damage tocartilage), seronegative arthropathies, diffuse idiopathic skeletalhyperostosis, post-arthroplasty, pressure ulcers, urinary tractinfections, arthrofibrosis, or glioma. The loaded particles areparticularly useful for locally treating in a confined space or regionof need such as surgical scars, severe burns, or cartilage repair.

In certain embodiments, the amount of agent loaded into themicroparticles may be from 0.1 ng to 10 mg, more particularly 100 ng to1000 μg, and most particularly, 1 to 100 μg agent per mg ofmicroparticles.

The microparticle system disclosed herein may provide for sustainedrelease of an agent. The agent release can be linear or non-linear(single or multiple burst release). In certain embodiments, the agentmay be released without a burst effect. For example, the sustainedrelease may exhibit a substantially linear rate of release of thetherapeutic agent in vivo over a period of at least 120 days, moreparticularly at least 60 days, and most particularly at least 20 days.By substantially linear rate of release it is meant that the therapeuticagent is released at a rate that does not vary by more than about 20%over the desired period of time, more usually by not more than about10%. It may be desirable to provide a relatively constant rate ofrelease of the agent from the delivery system over the life of thesystem. For example, it may be desirable for the agent to be released inamounts from 0.001 to 2 ng per day, more particularly 0.1 to 1 ng perday, for the life of the system. However, the release rate may change toeither increase or decrease depending on the formulation of the polymermicroparticle. The desired release rate and target drug concentrationcan vary depending on the particular therapeutic agent chosen for thedrug delivery system, and the subject's health.

The polymers for the microparticle may be bioerodible polymers so longas they are biocompatible. Preferred bio-erodible polymers arepolyhydroxyacids such as polylactic acid and copolymers thereof.Illustrative polymers include poly glycolide, poly lactic acid (PLA),and poly (lactic-co-glycolic acid) (PLGA). Another class of approvedbiodegradable polymers is the polyhydroxyalkanoates.

Other suitable polymers include, but are not limited to: polyamides,polycarbonates, polyalkylenes, polyalkylene glycols, polyalkyleneoxides, polyalkylene terephthalates, polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone,polyglycolides, polysiloxanes, polyurethanes and copolymers thereof,alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, celluloseesters, nitro celluloses, polymers of acrylic and methacrylic esters,methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose,cellulose acetate, cellulose propionate, cellulose acetate butyrate,cellulose acetate phthalate, carboxylethyl cellulose, cellulosetriacetate, cellulose sulphate sodium salt, poly(methyl methacrylate),poly(ethylmethacrylate), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexylmethacrylate),poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,polypropylene polyethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), poly(vinyl acetate), poly vinylchloride polystyrene, polyvinylpryrrolidone, alginate,poly(caprolactone), dextran and chitosan.

The percent loading of an agent may be increased by “matching” thehydrophilicity or hydrophobicity of the polymer to the agent to beencapsulated. In some cases, such as PLGA, this can be achieved byselecting the monomer ratios so that the copolymer is more hydrophilicfor hydrophilic drugs or less hydrophilic for hydrophobic drugs.Alternatively, the polymer can be made more hydrophilic, for example, byintroducing carboxyl groups onto the polymer. A combination of ahydrophilic drug and a hydrophobic drug can be encapsulated inmicroparticles prepared from a blend of a more hydrophilic PLGA and ahydrophobic polymer, such as PLA. The preferred polymer is a PLGAcopolymer or a blend of PLGA and PLA. The molecular weight of PLGA isfrom about 10 kD to about 80 kD, more preferably from about 10 kD toabout 35 kD. The molecular weight range of PLA is from about 20 to about30 kDa. The ratio of lactide to glycolide is from about 75:25 to about50:50. In one embodiment, the ratio is 50:50.

Illustrative polymers include, but are not limited to,poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 lactic acid to glycolicacid ratio, M_(n)=10 kDa, referred to as 502H);poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 lactic acid to glycolicacid ratio, M_(n)=25 kDa, referred to as 503H);poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 lactic acid to glycolicacid ratio, M_(n)=30 kDa, referred to as 504H);poly(D,L-lactic-co-glycolic acid) (PLGA, 50:50 lactic acid to glycolicacid ratio, M_(n)=35 kDa, referred to as 504); andpoly(D,L-lactic-co-glycolic acid) (PLGA, 75:25 lactic acid to glycolicacid ratio, M_(n)=10 kDa, referred to as 752).

In certain embodiments, the polymer microparticles are biodegradable.

The agent-loaded microparticles may have a volume average diameter of 10nm to 500 μm, more particularly 5 to 20 μm. The agent-loadedmicroparticles may be pore-less or they may contain varying amounts ofpores of varying sizes, typically controlled by adding NaCl during thesynthesis process.

The agent-loaded microparticle fabrication method can be single ordouble emulsion depending on the desired encapsulated agent solubilityin water, molecular weight of polymer chains used to make themicroparticles (MW can range from ˜1000 Da to over 100,000 Da) whichcontrols the degradation rate of the microparticles and subsequent drugrelease kinetics.

In some embodiments, the methods disclosed herein involve administeringto a subject in need of treatment a pharmaceutical composition, forexample a composition that includes a pharmaceutically acceptablecarrier and a therapeutically effective amount of the loadedmicroparticles disclosed herein. The compositions or loadedmicroparticles may be administered, for example, orally, parenterally(including subcutaneous injections (SC or depo-SC), intravenous (IV),intramuscular (IM or depo-IM), intrasternal injection or infusiontechniques), sublingually, intranasally (inhalation), intrathecally,topically, ophthalmically, or rectally. The pharmaceutical compositionmay be administered in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants, and/orvehicles. The loaded microparticles are preferably formulated intosuitable pharmaceutical preparations such as tablets, capsules, orelixirs for oral administration or in sterile solutions or suspensionsfor parenteral administration.

In some embodiments, one or more of the disclosed loaded microparticlesare mixed or combined with a suitable pharmaceutically acceptablecarrier to prepare a pharmaceutical composition. Pharmaceutical carriersor vehicles include any such carriers known to be suitable for theparticular mode of administration. Remington: The Science and Practiceof Pharmacy, The University of the Sciences in Philadelphia, Editor,Lippincott, Williams, & Wilkins, Philadelphia, Pa., 21^(st) Edition(2005), describes exemplary compositions and formulations suitable forpharmaceutical delivery of the compounds disclosed herein. In addition,the loaded microparticles may be formulated as the sole pharmaceuticallyactive ingredient in the composition or may be combined with otheractive ingredients.

Upon mixing or addition of the loaded microparticles to apharmaceutically acceptable carrier, the resulting mixture may be asolution, suspension, emulsion, or the like. Liposomal suspensions mayalso be suitable as pharmaceutically acceptable carriers. These may beprepared according to methods known to those skilled in the art. Theform of the resulting mixture depends upon a number of factors,including the intended mode of administration and the solubility of theloaded microparticles in the selected carrier or vehicle. Where theloaded microparticles exhibit insufficient solubility, methods forsolubilizing may be used. Such methods are known and include, but arenot limited to, using cosolvents such as dimethylsulfoxide (DMSO), usingsurfactants such as Tween®, and dissolution in aqueous sodiumbicarbonate. Derivatives of the inhibitors, such as salts or prodrugsmay also be used in formulating effective pharmaceutical compositions.The disclosed loaded microparticles may also be prepared with carriersthat protect them against rapid elimination from the body, such astime-release formulations or coatings. Such carriers include controlledrelease formulations, such as, but not limited to, microencapsulateddelivery systems.

The disclosed loaded microparticles and/or compositions can be enclosedin multiple or single dose containers. The loaded microparticles and/orcompositions can also be provided in kits, for example, includingcomponent parts that can be assembled for use. For example, one or moreof the disclosed compounds may be provided in a lyophilized form and asuitable diluent may be provided as separated components for combinationprior to use. In some examples, a kit may include a disclosed loadedmicroparticle and a second therapeutic agent for co-administration. Theloaded microparticles and second therapeutic agent may be provided asseparate component parts. A kit may include a plurality of containers,each container holding one or more unit dose of the loadedmicroparticles. The containers are preferably adapted for the desiredmode of administration, including, but not limited to tablets, gelcapsules, sustained-release capsules, and the like for oraladministration; depot products, pre-filled syringes, ampoules, vials,and the like for parenteral administration; and patches, medipads,creams, and the like for topical administration.

The loaded microparticles are included in the pharmaceuticallyacceptable carrier in an amount sufficient to exert a therapeuticallyuseful effect in the absence of undesirable side effects on the subjecttreated. A therapeutically effective concentration may be determinedempirically by testing the loaded microparticles in known in vitro andin vivo model systems for the treated disorder. In some examples, atherapeutically effective amount of the loaded microparticles is anamount that lessens or ameliorates at least one symptom of the disorderfor which the loaded microparticle is administered. Typically, thecompositions are formulated for single dosage administration. Theconcentration of active compound in the drug composition will depend onabsorption, inactivation, and excretion rates of the active compound,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art.

In some examples, about 0.1 mg to 1000 mg of a disclosed compound, amixture of such compounds, or a physiologically acceptable salt or esterthereof, is compounded with a physiologically acceptable vehicle,carrier, excipient, binder, preservative, stabilizer, flavor, etc., in aunit dosage form. The amount of active substance in those compositionsor preparations is such that a suitable dosage in the range indicated isobtained. The term “unit dosage form” refers to physically discreteunits suitable as unitary dosages for human subjects and other mammals,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect, in associationwith a suitable pharmaceutical excipient. In some examples, thecompositions are formulated in a unit dosage form, each dosagecontaining from about 1 mg to about 1000 mg (for example, about 2 mg toabout 500 mg, about 5 mg to 50 mg, about 10 mg to 100 mg, or about 25 mgto 75 mg) of the one or more compounds. In other examples, the unitdosage form includes about 0.1 mg, about 1 mg, about 5 mg, about 10 mg,about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 600mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, or more ofthe disclosed compound(s).

The disclosed loaded microparticles or compositions may be administeredas a single dose, or may be divided into a number of smaller doses to beadministered at intervals of time. The therapeutic compositions can beadministered in a single dose delivery, by continuous delivery over anextended time period, in a repeated administration protocol (forexample, by a multi-daily, daily, weekly, or monthly repeatedadministration protocol). It is understood that the precise dosage,timing, and duration of treatment is a function of the disease beingtreated and may be determined empirically using known testing protocolsor by extrapolation from in vivo or in vitro test data. It is to benoted that concentrations and dosage values may also vary with theseverity of the condition to be alleviated. In addition, it isunderstood that for a specific subject, dosage regimens may be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only.

The loaded microparticles are especially suitable for local and/orsustained delivery. For local delivery, the loaded microparticles areapplied directly to the tissue or organ for which treatment is sought.The effect of local delivery is limited primarily to the tissue or organto which the loaded microparticles are applied. For example, localdelivery may be accomplished through the use of compositions such asliniments, lotions, drops, ointments, creams, suppositories, emulsions,solutions, suspensions and the like. Local delivery can also beaccomplished using special delivery devices such as catheters, syringesor implantables designed to convey drug to a specific region in thebody.

The delivery composition may be a topical, syringable, or injectableformulation; and is suitable for local delivery of the active agent. Fortopical administration, the delivery composition is applied directlywhere its action is desired. Methods for topical delivery include theuse of ointments, creams, emulsions, solutions, suspensions and thelike. In other embodiments, the delivery composition is administered byapplication through a cannula, by injection, or as part of a lavage.Compositions for these types of local delivery can include solutions,suspensions and emulsions.

Examples of local administration include, but are not limited to,epicutaneous administration (i.e., application onto the skin);inhalation; as an enema for local administration to the bowel; ocular,for example, as eye drops for local administration to the conjunctiva;aural, for example, as ear drops; or intranasal. In other embodiments,an active agent can be administered locally from a device such as aballoon catheter. In another embodiment, local administration includesthe lavage of an open wound, the lavage containing delivery compositionsdescribed herein with antimicrobials or other wound healing medicaments.

In yet another embodiment, the loaded particles can be administered as acoating on an article for implantation. Such articles include polymerscaffolds containing stem/progenitor cells, stents, shunts, and thelike.

The loaded microparticles may also be delivered via an impregnated orcoated device such as a stent, for example, or an artery-insertedcylindrical polymer. The loaded microparticles may be administered, forexample, by local delivery from the struts of a stent, from a stentgraft, from grafts, or from the cover or sheath of a stent.

In some embodiments, the loaded microparticles are admixed with a matrixand formed into an implantable drug depot. Such a matrix may be apolymeric matrix. Polymeric matrices suitable for such use, include, forexample, lactone-based polyesters or copolyesters such as polylactide,polycaprolactonglycolide, polyorthoesters, polyanhydrides,polyaminoacids, polysaccharides, polyphosphazenes, poly (ether-ester)copolymers (e.g., PEO-PLLA); polydimethylsiloxane,poly(ethylene-vinylacetate), acrylate-based polymers or copolymers(e.g., polyhydroxyethyl methylmethacrylate, polyvinyl pyrrolidinone),fluorinated polymers such as polytetrafluoroethylene and celluloseesters. Suitable matrices may be nondegrading or may degrade with time,releasing the active compound.

The loaded microparticles can also be included in a hydrogel matrix foradministration to a subject, particularly for local and sustaineddelivery of the inhibitors disclosed herein.

In some embodiments, the pharmaceutical composition is formulated forinjection containing the loaded microparticles and a pharmaceuticalexcipient suitable for injection. The forms in which the compositionsmay be incorporated for administration by injection include aqueous oroil suspensions, or emulsions, with sesame oil, corn oil, cottonseedoil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterileaqueous solution, and similar pharmaceutical vehicles.

Aqueous solutions in saline are also conventionally used for injection.Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and thelike (and suitable mixtures thereof), cyclodextrin derivatives, andvegetable oils may also be employed. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, forthe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like.

Sterile injectable solutions are prepared by incorporating the loadedmicroparticles in the required amount in the appropriate solvent withvarious other ingredients as enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, certaindesirable methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Another exemplary formulation for use in the methods disclosed hereinemploys transdermal delivery devices (“patches”). Such transdermalpatches may be used to provide continuous or discontinuous infusion ofloaded microparticles in controlled amounts, either with or withoutanother agent.

In certain embodiments, the loaded microparticles may be administered ina scaffold, for instance filling a defect in cartilage (it could be apolymeric scaffold or a hydrogel) or by covering a burn with a smear ofgelatin which has the particles inside or of fibrin glue or with amembrane patch. Transdermal microneedles could also be a way ofadministering the microparticles as well as skin patches.

Examples

Dorsomorphin Microparticle Fabrication

Reagents:

-   -   4.2 kDa 50:50 PLGA    -   DI H₂O    -   Dichloromethane (DCM)    -   4% Polyvinyl Alcohol (PVA)

-   1) Surfactants: make up 2% (60 ml/100 ml beaker: 30 ml of PVA in 30    ml of DI H₂O) and 1% PVA solutions (80 ml/in 250 ml beaker: 20 ml of    PVA in 60 ml of DI H₂O).

-   2) Prepare 0.5 ml of 0.5 mg/ml dorsomorphin in DMSO.

-   3) Prepare 5% PLGA solution (200 mg PLGA; 4 ml DCM).

-   4) Combine dorsomorphin and PLGA solutions.

-   5) Place the 100 ml beaker of 2% PVA under the homogenizer & the 250    ml beaker of 1% PVA on magnetic stirrer, spinning at 500 rpm.

-   6) Pour dorsomorphin/PLGA solution into the 2% PVA solution that is    being homogenized at 2700 rpm.

-   7) Homogenize for 1 min at 2700 rpm.

-   8) Immediately pour into 1% PVA solution that is being stirred at    500 rpm.

-   9) Stir for 3 hours to let DCM evaporate, after which remove and    wash in D.I. water 4 times.

-   10) Spin/wash with DI H₂O in 50 ml falcon tubes to remove PVA. Spin    at 1000 rpm for 5 minutes then decant.

-   11) Repeat spin/wash at least 3 times.

-   12) Collect/resuspend microparticles in less than 5 ml DI H₂O and    place in scintillation vial (weight vial beforehand)

-   13) Freeze in −80 C freezer

-   14) Transfer in the lyophilizer and keep it there for 2-3 days.    T3 Induces Hypertrophy Primary Chondrocytes Hypertrophy

In autologous chondrocytes implantation (ACI) chondrocytes are implantedin a local cartilage defect. In an in vitro model of ACI based on asmall osteochondral unit, as the one we use, a central cartilage defectwould be filled with chondrocytes in a hydrogel. To simulate conditionsof calcification, the chondrocytes will be pre-cultured in a hypertrophyinducing medium. After seeding, both native cartilage and implantedchondrocytes would be exposed to chondrogenic medium. We have previouslyshown that T3 induces hypertrophy in MSCs that underwent chondrogenicdifferentiation. Here we show that T3 is effective to induce primarychondrocytes hypertrophy that is maintained even after exposure tochondrogenic medium (see FIGS. 7A-7F). Chondrogenic medium andosteogenic medium are used as comparison.

A Calcification Model

A calcification model was developed inducing hypertrophy of humanchondrocytes (monolayer culture and 3D photocrosslinkable gelatinconstructs) or osteochondral (OC) tissue plugs by exposure to thehormone tri-iodothyronine (T3). Human osteoblasts were used as apositive control. Both chondrocytes, and osteoblasts (cells andconstructs) were treated with 3 different conditions for eachexperiment: Chondrogenic Medium (CM), Osteogenic Medium (OM), andHypertrophic Medium (HM).

For the 3D gelatin constructs, chondrocytes were harvested from the kneeof a 65-year old male donor and osteoblasts from the hip of a 65-yearold male donor. After 14 days and 21 days, 1 group of constructs in boththe positive control and the experimental group were switched tochondrogenic media until the end of the 28-day experiment. Another groupof constructs for each condition was carried out for the total 28 days.Histology and RT-PCR (COL1, COL2, ACAN, ALP, RUNX-2, COL10, OCN, MMP13)were performed to confirm the presence or absence of hypertrophy. Theresults are shown in FIGS. 8A-8I.

In Vivo Heterotopic Ossification after MPC and BMP2 Injection

Heterotopic ossification (HO), the formation of mature bone in the softtissues, is a frequent complication following trauma. The pathogenesisof HO is not well understood, but is hypothesized to involve aninappropriate cellular response to the inflammatory environment andspecific osteogenic signals therein. We have previously isolated andcharacterized a population of mesenchymal progenitor cells (MPCs)derived from human blast-traumatized muscle (BDM-MSC) that may representthe cellular component of the disease.

Formation of ectopic bone nodules in the hind limbs of SCID miceinjected with MPC+/−BMP2 as compared to those injected with humanforeskin fibroblasts (HFF)+/−BMP2 is shown in FIG. 9. Bone volume isdetermined by μCT. Injection of collagen served as a negative control.

These results indicate that BMP induces significantly more osteogenesisby MPCs than HFF. BMP alone induces osteogenesis at levels comparable tothat measured in MPC+BMP injected samples at week 2 and 4 afterinjection. However, HO lesions induced by BMP alone were significantlyreduced after 6 weeks as compared to samples with MPCs present,indicating that the MPCs are playing a substantial role in theproduction and maintenance of the HO lesions.

Inhibition of muscle-derived multipotent progenitor cells (MPC)osteogenesis in 2D by dorsomorphin (DM) (2.5 mM) as shown by alizarinred staining (Mag=10×) is shown in FIG. 10. MPCs from the same donorwere plated and cultured in different conditions. Controls received onlyregular growth medium, the BMP+ group were exposed to medium additionedwith BMP which is a strong inducer of osteogenesis, the DM+ groups wereexposed to medium additioned with soluble dorsomorphin to control foreffects of dorsomorphin alone, and the test group BMP+DM+ was exposed tomedium containing both soluble BMP and soluble dorsomorphin. Media wererenewed twice a week. Cells were assessed for osteogenesis/calcificationat 1, 3, 5, and 7 weeks. Osteogenesis/deposition of calcium was assessedby alizarin red staining, a common histological staining procedure. Thecontrol group exhibited a moderate tendency to calcification evenwithout BMP stimulation, as indicated by the progressive reddening overtime. Exposure to BMP induced a strong osteogenic/calcificationphenotype, marked by the strong red staining especially at 7 weeks.Dorsomorphin alone seemed to inhibit even the natural tendency of bonemarrow-derived MSCs to calcify. When both BMP and dorsomorphin wereadded to the media, no calcification happened, indicating thatdorsomorphin is able to inhibit calcification even when it is potentlyinduced by BMP as would occur in vivo.

Inhibition of BMD-MSC osteogenesis in 2D culture by dorsomorphin (DM) isshown in FIG. 11. Phase contrast images of alizarin red stained culturestreated with BMP (B), DM, and BMP+DM (D) as compared to control (A) andDM (C) cultures. The test was performed as the one in FIG. 10 but usingbone marrow-derived MSCs and with a sole time point of 21 days. In thiscase as well, BMP exposure induced a strong calcium deposition, whichwas completely inhibited by the addition of soluble dorsomorphin insolution.

Formation of ectopic bone nodules in the hind limbs of SCID miceinjected with MPC+BMP2+/−DM. DM was delivered in solution or in microparticles. The DM was loaded into PLGA microparticles. Bone volume isdetermined by μCT.

Micro-CT 3D reconstructions HO lesions in vivo following injection of(A) MPC/BMP2+DM in microparticles (mp), (B) MPC/BMP2+ soluble (s) DM,and (C) MPC/BMP2 alone. All scans were normalized to the device hydroxyapatite standard (in vivo μCT 40; ScanCo).

These results shown in FIG. 12 indicate that the injection of 25 ng/mlBMP induces MPC osteogenesis within the muscle tissue. The co-injectionof microparticles loaded with DM inhibited this BMP2-induced MPCheterotopic ossification. sDM stands for soluble dorsomorphin, that isdorsomorphin that is solely injected with the cells in the mouse limb.sDM rapidly diffuses away or is all metabolized by cells in a shorttime, and it then has no long term effect on the locally injected cells.Mp DM stands for microparticles containing dorsomorphin. As theparticles degrade, dorsomorphin is released locally and the injectedcells are continually exposed to dorsomorphin coming in low overalldoses but sufficient local concentrations to induce a long term effectson injected cells that do not calcify (hardly any heterotopic bonevolume is visible). A microparticles release system is then necessaryfor dorsomorphin to be effective. The microparticles were loaded asdescribed in the previous protocol and are particles from the same batchfrom which particles were taken to perform the release profile in FIG.4.

In view of the many possible embodiments to which the principles of thedisclosed embodiments may be applied, it should be recognized that theillustrated embodiments are only examples and should not be taken aslimiting the scope of the invention.

What is claimed is:
 1. A therapeutic scaffold comprising (i) BMP Iinhibitor-loaded microparticles and (ii) chondrocytes.
 2. The scaffoldof claim 1, wherein the inhibitor is dorsomorphin.
 3. The scaffold ofclaim 1, wherein the inhibitor is LDN-193189, SB505124, noggin, cordin,or gremlin.
 4. The scaffold of claim 1, wherein the inhibitor is acompound, or a pharmaceutically acceptable salt thereof, represented byformula I:

wherein X is selected from CR¹⁵ and N; Y is selected from CR¹⁵ and N; Zis selected from CR³ and N; Ar is selected from substituted orunsubstituted aryl and heteroaryl; L₁ is absent or selected fromsubstituted or unsubstituted alkyl and heteroalkyl; A and B,independently for each occurrence, are selected from CR¹⁶ and N; E andF, independently for each occurrence, are selected from CR⁵ and N; R³represents a substituent; R⁴ is selected from substituted orunsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, acyl, carboxyl, ester, hydroxyl,alkoxyl, alkylthio, acyloxy, amino, acylamino, carbamate, amido,amidino, sulfonyl, sulfoxido, sulfamoyl, or sulfonamido; R⁵,independently for each occurrence, represents a substituent selectedfrom H and substituted or unsubstituted alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, aralkyl, heteroaryl,heteroaralkyl, cycloalkylalkyl, heterocyclylalkyl, halogen, acyl,carboxyl, ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino,acylamino, carbamate, amido, amidino, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamido, or two occurrences of R⁵ taken together withthe atoms to which they are attached form a substituted or unsubstituted5- or 6-membered cycloalkyl, heterocycloalkyl, aryl, or heteroaryl ring;R¹³ is absent or represents 1-2 substituents on the ring to which it isattached and, independently for each occurrence, is selected fromsubstituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, halogen, hydroxyl,alkoxyl, alkylthio, acyloxy, acylamino, carbamate, cyano, sulfonyl,sulfoxido, sulfamoyl, or sulfonamido; R¹⁵, independently for eachoccurrence, represents a substituent, selected from H and substituted orunsubstituted alkyl, heteroalkyl, cycloalkyl, heterocyclyl,cycloalkylalkyl, heterocyclylalkyl, halogen, hydroxyl, alkoxyl,alkylthio, acyloxy, acylamino, carbamate, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamido; and R¹⁶, independently for each occurrence,represents a substituent selected from H and substituted orunsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, aralkyl, cycloalkyl,heterocyclyl, aryl, heteroaryl, heteroaralkyl, cycloalkylalkyl,heterocyclylalkyl, halogen, acyl, carboxyl, ester, hydroxyl, alkoxyl,alkylthio, acyloxy, amino, acylamino, carbamate, amido, amidino, cyano,sulfonyl, sulfoxido, sulfamoyl, or sulfonamido.
 5. The scaffold of claim1, wherein the inhibitor is a compound, or a pharmaceutically acceptablesalt thereof, represented by formula II:

wherein X is selected from CR¹⁵ and N; Y is selected from CR¹⁵ and N; Zis selected from CR³ and N; Ar is selected from substituted orunsubstituted aryl and heteroaryl; L₁ is absent or selected fromsubstituted or unsubstituted alkyl and heteroalkyl; Py is substituted orunsubstituted 4-pyridinyl or 4-quinolinyl; R³ represents a substituentselected from H and substituted or unsubstituted alkyl, heteroalkyl,cycloalkyl, halogen, hydroxyl, alkoxyl, alkylthio, acyloxy, acylamino,carbamate, cyano, sulfonyl, sulfoxido, sulfamoyl, or sulfonamido; R⁴ isselected from substituted or unsubstituted alkyl, alkenyl, alkynyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, acyl, carboxyl,ester, hydroxyl, alkoxyl, alkylthio, acyloxy, amino, acylamino,carbamate, amido, amidino, sulfonyl, sulfoxido, sulfamoyl, orsulfonamido; R¹³ is absent or represents 1-2 substituents on the ring towhich it is attached and, independently for each occurrence, is selectedfrom substituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocyclyl, cycloalkylalkyl, heterocyclylalkyl, halogen, hydroxyl,alkoxyl, alkylthio, acyloxy, acylamino, carbamate, cyano, sulfonyl,sulfoxido, sulfamoyl, or sulfonamido; and R¹⁵, independently for eachoccurrence, represents a substituent selected from H and substituted orunsubstituted alkyl, heteroalkyl, cycloalkyl, heterocyclyl,cycloalkylalkyl, heterocyclylalkyl, halogen, hydroxyl, alkoxyl,alkylthio, acyloxy, acylamino, carbamate, cyano, sulfonyl, sulfoxido,sulfamoyl, or sulfonamide.
 6. The scaffold of claim 1, wherein themicroparticles comprise poly glycolide, poly lactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), or a combination thereof.
 7. Thescaffold of claim 6, wherein the loaded microparticles have a volumeaverage diameter of 5 to 20 μm.
 8. The scaffold of claim 7, wherein theinhibitor is dorsomorphin.
 9. The scaffold of claim 1, wherein theloaded microparticles have a volume average diameter of 10 nm to 500 μm.10. The scaffold of claim 1, wherein the loaded microparticles have avolume average diameter of 5 to 20 μm.
 11. The scaffold of claim 1,further comprising mesenchymal stem cells.
 12. The scaffold of claim 1,wherein the scaffold is a stent or shunt.
 13. The scaffold of claim 1,wherein the BMP I inhibitor-loaded microparticles and the chondrocytesare located on a surface of the scaffold.